携带猪流行性腹泻病毒S基因的减毒鼠伤寒沙门菌的构建与鉴定  

Construction and identification of attenuated Salmonella typhimurium harboring S gene of porcine epidemic diarrhea virus

作　　者：梁恩涛 廖晓丹[1] 黄小波[1,2] 文心田[1,2] 曹三杰[1,2] 阳酉萍 段素芬[1] 张丹[1] 张小会[1] 

机构地区：[1]四川农业大学动物医学院动物传染病与基因芯片实验室,四川雅安625014 [2]四川农业大学人兽共患病研究室与猪病防治研究中心,四川成都611130

出　　处：《中国兽医科学》2014年第3期264-270,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31072144;30901084);国家公益性行业(农业)科研专项(20123056)

摘　　要：为构建可有效抵抗猪流行性腹泻病毒(PEDV)入侵的诱导黏膜免疫反应的核酸疫苗,采用RTPCR方法扩增了PEDV SC-L株的S基因主要抗原编码区域(简称S1),插入pMD19-T载体,构建载体pMD19-T-S1,再将S1基因片段插入双启动子真核表达载体pVAXD中,构建了pVAXD-S1表达载体。经免疫荧光法鉴定S1基因可在COS-7细胞中正常表达后,将pVAXD-S1电转入减毒鼠伤寒沙门菌SL7207中,构建了携带S1基因的重组减毒沙门菌SL7207(pVAXD-S1),并对该重组菌株的生长曲线、携带质粒的稳定性、口服小鼠的安全性及目的基因在体内转录等特性进行了鉴定。结果显示,SL7207(pVAXD-S1)在含卡那霉素(100μg/mL)的培养环境中稳定性良好,以每只1×109 CFU口服免疫BALB/c小鼠具有良好的安全性,携带的S1基因能在小鼠回肠末端组织内正常转录及表达。本研究为深入开展减毒沙门菌疫苗菌株SL7207(pVAXD-S1)的免疫评价及构建PEDV与其他抗原基因联合免疫DNA疫苗奠定了基础。To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus （PEDV） infection,the PEDV SC-L strain S gene＇s S1 region（S1 gene, 2 367 bp, encoding amino acid 1-- 789 of N-terminal S protein） was amplified by RT-PCR and inserted into pMD19-T to construct pMD19-T- S1. Then the S1 gene fragment was inserted into dual-promoter eukaryotic expression vector pVAXD to construct pVAXD-S1. After the expression of S1 gene was proved by indirect immunofluorescence assay in COS-7 cell,and pVAXD-S1 was electrotransformed into the attenuated Salmonella typhimurium SL7207. The plasmid stability and safety, and transcription analysis of recombinant Salmonella SL7207 （pVAXD- S1） were proved. SL7207 （pVAXD-S1） was steady in the culture environment within 100μg/mL kanamycin and safe to BALB/c mice at the dosage of 1× 109 CFU by oral administration. In addition,the S1 gene could be transcribed in the ileum tissue of the mouse immunized with SL7207 （pVAXD-S1）. The results showed that the study laid the foundation for evaluating the immunogenicity of SL7207 （pVAXD-S1） and for con- struction of DNA vaccine harboring PEDV S gene and other immunodominant genes.

关 键 词：猪流行性腹泻病毒 S基因 减毒鼠伤寒沙门茵 构建 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]